Transcript Raman microspectroscopy of oral bacteria

Medical adventures in the
near-infrared
What’s Up In the Biomedical
Spectroscopy Lab
Andrew Berger
The Institute of Optics
October 11, 2007
Participants and Collaborators
• Biomedical Spectroscopy Laboratory members:
Rolf Saager, Zach Smith, Brooke Chuzles,
• Brain and Cognitive Sciences: Prof. Richard Aslin,
Dr. Andrea Gebhardt
• Dept. of Imaging Sciences: Prof. Tom Foster
• Center for Oral Biology: Prof. Rob Quivey
• Dept. of Immunology: Prof. Tim Mosmann,
Dr. Sally Quaetert
• Dept. of Pathology: Prof. Bob Mooney
Outline
• The big picture: biomedical optics and
the near infrared
• Elastic scattering: photon diffusion
• cerebral monitoring
• Inelastic scattering: Raman spectroscopy
• various projects
The grand scheme of things
Biomedical
Optics
alteration
manipulation
observation
The grand scheme of things
absorption
Biomedical
Optics
elastic scattering
polarization
fluorescence
inelastic scattering
observation
spectroscopy
vibrational
electronic
courtesy V. Venugopalan, http://www.osa.org/meetings/archives/2004/BIOMED/program/#educ
rotational
DNA
biological
window
The near-infrared “window”
Outline
• The big picture: biomedical optics and
the near infrared
• Elastic scattering: photon diffusion
• infant brain monitoring
• Inelastic scattering: Raman spectroscopy
• various projects
Rolf Saager
?
vision
hearing
speech
motor/sensory
Using light to study blood
Absorptivity of Pure Blood ()
two
measurements:
absorption per
unit length at
two wavelengths
690 nm
  a690 nm    HbO
2
 830 nm    830 nm
 
 
 a
  HbO 2
two
unknowns:
oxy- and deoxyhemoglobin
concentrations
 H690 nm  HbO2 


830 nm 
 Hb  Hb 
Noninvasive monitoring of
hemodynamics
optical power
measurements
increased blood supply
heartbeat
oxy and deoxy
hemoglobin
concentration
changes
light in (690, 830 nm)
light collected
A problem
Measurement sensitive to
both brain and scalp
detector
A solution?
second
detector
Measurement sensitive to
both brain and scalp
detector
Add a second detector that
senses only the scalp
Subtract signals to reveal
brain-only features?
System Layout
1-10 kHz Modulation
830 Laser
Analog Out
DAQ Card
Source 1
High Speed
DAQ Card
690 Laser
830 Laser
APDs
Source 2
near
near
far
far
far
far
far
far
690 Laser
Decoded Wavelength Data
l830
l690
Sample
Oh, yeah: Reality
A physicist’s head
Scalp hemodynamics
Cerebral Hemodynamics
A real head
Our data…
Other people’s raw data…
large separation
short separation
residual
raw data
courtesy of
Joseph Culver,
Washington
University
Bring the kids, too!
Outline
• The big picture: biomedical optics and
the near infrared
• Elastic scattering: photon diffusion
• infant brain monitoring
• Inelastic scattering: Raman spectroscopy
• multiple projects
• Other projects
Zach Smith
Brooke Chuzles
Raman scattering
incident photon
with energy E
molecule
Raman scattering
incident photon
with energy E
molecule gains energy E
scattered photon has
energy E -E
to
detector
853
813
667
1211
1127
Raman shift (cm-1)
amide I
1651
1259
1092
1340
RNA bases
1580
902
720
aromatic amino acids
C-H 2 def. 1457
amide III
C-N, C-C str.
phenylalanine 1005
tyrosine
cytosine, uracil 783
adenine
guanine
619 phenylalanine
intensity (arb. units)
Raman Spectrum of a Cell
Neutrophil
Peripheral blood mononuclear cell
Angularly-resolved scattering
d
collimated
incident
light
angular distribution
dependent upon size
and refractive index
mismatch
n1
n2
Experimental System
Angle Mapped to Position
Weak
in Fourier plane
M
CCD
Inverse telescope
4% reflector
CCD
TV
confocal
90° periscope Relay Lens pinhole OD 3 NDF
Semrock NF
100 mW
785nm laser
Multi-mode
fiber
Spatial filter BPF
λ/2
P
S
Scattering versus angle for sphere of diameter d = 4.7um, m = 1.1955
beamwaist = 2.59e-006
137
151
166
180

195
209
224
137
151
166
180

195
209
224
Raman spectrum
angular scattering
pattern
monocyte
wavenumber shift
neutrophil
urea
Urine sample
Amide I
C-H bending
Amide III
C-C stretching
phenylalanine
C-O-C stretching
tyrosine
tyrosine
Phenylalanine
tyrosine
Blood serum sample
Raman spectroscopy of biofluids
T = 150 seconds
T = 60 seconds
Blood serum: total protein, albumin
and globulin
71 samples
albumin
globulin
TP
Urine: UUN (urine urea nitrogen) and
creatinine
UUN
creatinine
Biologically speaking...
Streptococcus sanguis
(S. sanguis)
Streptococcus mutans
(S. mutans)
HIGH SUGAR DIET
LACTIC ACID
Quantitative Raman measurement of S. mutans
concentration in 3-species mixtures
Predicted S. mutans concentration
100%
80%
Calibration set
Validation set
Error of calibration: 6.8%
Error of validation: 6.7%
60%
40%
20%
0
0
20% 40% 60% 80% 100%
Reference S. mutans concentration
Project goal:
perform similar
quantitative analysis
of biofilms and
plaque specimens
("identify and quantify
the bad guys that lead
to tooth decay")
Biomedical spectroscopy lab information:
AJB office:
Goergen Hall 405, 273-4724, ajberger@optics...
labs:
Goergen Hall 433, 435, 436